Nucleic acid nanotube liquid crystals and use for NMR structure determination of membrane proteins

ABSTRACT

Compositions and methods for preparing nucleic acid nanotubes using DNA origami techniques are described, which provide for nanotubes of predictable and uniform length. The nucleic acid nanotubes thus formed are suitable as liquid crystal preparations enabling liquid-crystal NMR spectroscopy of proteins solubilized in detergent.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/793,788, filed Apr. 21, 2006, and U.S.Provisional Application Ser. No. 60/904,266, filed Feb. 28, 2007.

TECHNICAL FIELD

The invention generally relates to nucleic acid nanotubes. Moreparticularly the invention relates to compositions and methods formaking nucleic acid nanotubes that are suitable for performingliquid-crystal NMR spectroscopy of detergent-solubilized membraneproteins.

BACKGROUND

Structure determination of membrane proteins is an important challengefor biomedical science. About thirty percent of expressed proteins spanlipid bilayers, yet structures of only about one hundred membraneproteins have been resolved. Membrane proteins are encoded by 20-35% ofgenes but represent fewer than one percent of known protein structuresto date. Knowledge of their structures will be enormously insightful forcell biology. Furthermore, membrane proteins are important as drugtargets. The slow rate of membrane-protein structure determinationrepresents a significant bottleneck for both basic and appliedbioscience discovery. This bottleneck largely derives from difficultiesin forming well-ordered three-dimensional crystals of membrane proteins.Solution NMR presents an attractive alternative for the study ofmembrane proteins, as high-resolution structural information can beobtained for proteins up to 80 kD in size without the need forcrystallization. Residual dipolar couplings (RDC's), commonly measuredfor biological macromolecules weakly aligned by liquid-crystallinemedia, are important global angular restraints for NMR structuredetermination. For membrane proteins greater than 15-kDa in size,Nuclear-Overhauser-effect (NOE)-derived distance restraints aredifficult to obtain, and RDC's could serve as the main reliable sourceof NMR structural information. In many of these cases, RDC's wouldenable full structure determination that otherwise would be impossible.However, none of the existing liquid-crystalline media used to alignwater-soluble proteins are compatible with the detergents required tosolubilize membrane proteins.

For solution NMR, macromolecules must be solubilized in water tofacilitate fast tumbling; the faster the tumbling, the better thespectra. To promote water solubility, membrane proteins must becomplexed with detergent micelles. The micelle-protein complex isconsiderably larger than the protein alone, and tumbling is relativelyslow as a result. This increase in effective size is especiallyproblematic for α-helical membrane proteins greater than 15 kD in size,where resonance peaks are closely spaced and become irresolvable withthe fast coherence relaxation of slowly tumbling macromolecules. Inorder to obtain information about the internuclear angles, each proteinmust be made to tumble in a weakly ordered regime. The appropriate weakordering, about 0.1%, can be achieved by dissolving the protein in anappropriate concentration of a suitable alignment material. For example,water-soluble proteins can be aligned weakly by a suitable amount with˜1.5-2% Pfl filamentous phage, which forms a liquid crystal at thatconcentration. The easiest method for weak alignment of proteins isthrough mixing the protein with a liquid-crystalline medium, such as Pflfilamentous phage, DMPC/DHPC bicelles, C12E5 polyethylene glycol, orcellulose crystallites. However, none of these media are compatible withdetergent-solubilized membrane proteins.

The general applicability of solution NMR spectroscopy to structuralcharacterization of intact α-helical membrane proteins has beendemonstrated by the structure determination of the 15-kDa Mistic proteinand the 30-kDa pentameric phospholamban, as well as the completeassignment of backbone resonances and secondary structures of the 44-kDatrimeric diacylglycerol kinase and the 68-kDa tetrameric KcsA potassiumchannel. Despite such progress, full-scale structure determination ofα-helical membrane proteins remains challenging and rare. Due to thelarge fraction of methyl-bearing residues in membrane proteins and tothe added molecular weight of detergent micelles, the low chemical-shiftdispersion of α-helical proteins is obscured by resonance overlap andline broadening, making assignment of side-chain methyl resonancesextremely difficult. Without side-chain chemical shifts, it isimpossible to obtain a sufficient number of long-range NOE-deriveddistance restraints for folding secondary segments into the correcttertiary structure. Therefore, development of alignment media foraccurate RDC measurements from α-helical membrane proteins would enhancesignificantly the capability of solution NMR in structure determinationof this important class of targets.

The most effective method for weak alignment involves mixing the proteinof interest with large particles that form stable liquid crystals at lowconcentration (˜1.5-5% w/v). Liquid crystals that have been used toalign water-soluble proteins include DMPC/DHPC-bicelle liquid crystals,filamentous phage particles, ternary mixtures of cetylpyridinium Cl/Br,hexanol, and sodium Cl/Br, binary mixtures of polyethylene glycol andhexanol, and cellulose crystallites. However, none could be applied tomembrane proteins due to incompatibility with the zwitterionic oranionic detergents typically used to solubilize membrane proteins forstructural study. The only method currently available for weak alignmentof membrane proteins involves the use of strained (radially or axiallycompressed) polyacrylamide gels. However, dissolving protein-micellecomplexes to high concentration in gels is notoriously difficult due tothe inhomogeneous pore size of randomly cross-linked gel matrices. Thusthe measured RDC's are of limited accuracy.

Nucleic acid nanotube liquid crystals can extend the advantages of weakalignment to NMR structure determination of a broad range ofdetergent-solubilized membrane proteins. Alignment media comprised of800 nm heterodimer DNA nanotubes should be broadly useful for providingglobal structural restraints in solution NMR studies of membraneproteins. As a large number of helical membrane proteins of greatbiomedical interest are between 20-30 kDa in size—well below the currentsize limitation of solution NMR spectroscopy—new experimental systemsfor obtaining NMR structural information in the presence of detergentsare of fundamental importance. DNA nanotechnology, which affordsversatile molecular design and sub-nanometer-scale precision, has beenpursued as a route towards building host lattices to position guestmacromolecules for crystallographic structural studies. The presentinvention employs solution NMR instead of crystallographic methods, andvalidates the potential of DNA nanotechnology for imposing order ontarget macromolecules to acquire atomic-resolution structuralinformation.

SUMMARY OF THE INVENTION

The invention is related to novel compositions and methods for preparingliquid crystalline solutions of nucleic acid nanotubes suitable forperforming liquid-crystal NMR spectroscopy of proteins, includingdetergent-solubilized membrane proteins. By virtue of being constructedfrom nucleic acids, these nanotubes generally are resistant todetergents, and can be constructed, for example, to mimic the shape andsize of filamentous phage particles.

It is an object of the invention to provide a composition comprisingnucleic acid nanotubes having uniform length. Each nanotube comprises aplurality of linked double-stranded nucleic acid helices, and eachnanotube is formed from at least one single-stranded scaffold nucleicacid molecule and a plurality of staple oligonucleotides. The nucleicacid can be DNA. The nucleic acid nanotubes can form aliquid-crystalline phase in solution, and proteins solubilized indetergent can be aligned weakly using the nanotube liquid crystals.

A further object of the invention is to provide nucleic acid nanotubesin which the average length of the nucleic acid nanotubes is given bythe length of the single-stranded scaffold nucleic acid divided by thenumber of double stranded nucleic acid helices comprising each nanotube.In some embodiments the length of the helices comprising the nucleicacid nanotubes varies by no more than 20% of the average length of thehelices, and in certain embodiments by no more than 10% of the averagelength of the helices. The length of the nucleic acid nanotubes isgreater than about 50 nanometers, and about 400 nanometers. Thenanotubes comprise at least 3 adjacent double-stranded helices. Incertain embodiments the nanotubes consist of 5, 6, or 7 adjacenthelices. In other embodiments, DNA heterodimer nanotubes 800 nanometersin length can be constructed from 400 nanometer monomers of two types,one type of monomer self-assembling with the second type of monomer.

A further object of this invention is to provide a method of preparingnucleic acid nanotubes. The method comprises preparing a solutioncomprising a single-stranded scaffold nucleic acid and a plurality ofstaple oligonucleotides, heating the solution to denature the scaffoldnucleic acid and staple oligonucleotides, and cooling the solution toroom temperature. The nucleic acid nanotubes thus formed will have auniform length.

A further object of this invention is to provide a method to performliquid-crystal NMR spectroscopy of proteins using nucleic acidnanotubes. The method comprises suspending nucleic acid nanotubes in asolution, forming a liquid crystalline phase comprising the nucleic acidnanotubes, adding a protein to the solution, performing NMR spectroscopyon the protein and nucleic acid nanotube mixture. In some embodiments,the protein added to the solution is solubilized in detergent. In otherembodiments, the protein is a membrane protein solubilized in detergent.In other embodiments, the protein is present at a concentration of atleast 0.1 mM.

Further features and advantages of the invention and further embodimentswill become more fully apparent in the following description of theembodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows various representations of a 800 nm DNA nanotubeheterodimer.

FIG. 1 a is a stylized 3-dimensional representation highlighting the 14nm segment that forms the junction between the two 400 nm monomers.

FIG. 1 b is a segment diagram in which each monomer consists of 28segments of length 42 base pairs, as well as a head and tail segment oneach end.

FIG. 1 c is a schematic of the two scaffold strands of each monomer(without the complementary staple oligonucleotides), each monomerconsisting of a modified M13 bacteriophage single-stranded DNA genome oflength 7308 bases.

FIG. 1 d is a cross-sectional schematic view of the DNA nanotube shownin

FIG. 1 a.

FIG. 1 e is a schematic of the 14 nm junction between the linkednanotube monomers, showing the 42 base pair link between the twoscaffold strands and their complementary staple strands.

FIG. 1 f is a schematic of a typical 42 base pair segment of thecompleted nanotube, showing that a crossover pattern consisting of sixstaple strands repeats itself every 42 base pair segment along thelength of the nanotube.

FIG. 2A is a schematic representation of a six-helix bundle DNAnanotube. FIG. 2B is a representation of the arrangement of scaffoldstrand and staple oligonucleotides of the six-helix DNA nanotube of FIG.2A. The dark line represents the uninterrupted scaffold DNA, folded intoa six-strand arrangement, with a seam formed between helix 2 and helix3, and another seam between helix 4 and helix 5. The stapleoligonucleotides are generally 42 bases long, each contributing three14-base segments, one to each of three adjacent portions of the scaffoldDNA.

FIG. 3 shows schematic representations of the front and rear overhangsof the pre-dimerization monomers that combine to form DNA heterodimernanotubes

FIGS. 3 a and 3b show, respectively the “capped” scaffold-plus-stapleshead, and the unpaired scaffold and staple strands of the tail of thefront monomer.

FIGS. 3 c and 3d show, respectively, the unpaired scaffold and staplesof the head, and the “capped” scaffold-plus-staples tail of the rearmonomer.

FIG. 3 e shows the inter-monomer junction of a DNA heterodimer nanotube,in which the unpaired scaffold and staples of the front monomer arecomplementary to and join with the unpaired staples and scaffold of therear monomer.

FIGS. 4A-4D shows a Python computer program script for the sequencedetermination of the components of six-helix bundle DNA nanotubes.

FIGS. 5A-5B is the Python computer program script used to generate frontmonomer core oligonucleotides and head caps.

FIGS. 6A-6B is the Python computer program script used to generate rearmonomer core oligonucleotides and tail caps.

FIGS. 7A-7B is the Python computer program script used to generate frontmonomer tail connector oligonucleotides and rear head connectoroligonucleotides.

FIG. 8 shows the sequences used in the example for front monomer headcap staples of a six-helix bundle DNA nanotube (SEQ ID NOS 1-3,respectively, in order or appearance).

FIG. 9 shows the sequences used in the example for front monomer tailconnector staples of a six-helix bundle DNA nanotube (SEQ ID NOS 4-6,respectively, in order or appearance).

FIG. 10 shows the sequences used in the example for rear monomer headconnector staples of a six-helix bundle DNA nanotube (SEQ ID NOS 7-9,respectively, in order or appearance).

FIG. 11 shows the sequences used in the example for rear monomer tailcap staples of a six-helix bundle DNA nanotube (SEQ ID NOS 10-13,respectively, in order or appearance).

FIGS. 12A-C is the sequence of the M13 mp 18 derived single stranded DNAscaffold used in the DNA nanotube of FIG. 2 (SEQ ID NO: 14).

FIGS. 13A-D lists the sequences of each of the staple oligonucleotidesthat build to the scaffold DNA used in the DNA nanotube of FIG. 2 (SEQID NOS 15-195, respectively, in order or appearance).

FIG. 14 shows a computer-generated random 59-base sequence inserted intoM13 mp 18 at insert position 6258 to generate recombinant M13filamentous bacteriophage (SEQ ID NO: 196).

FIG. 15 shows the sequences that were used in the example to constructthe M13 mp 18 insert fragment of FIG. 14, together with flanking regions(109 base pairs total) (SEQ ID NOS 197-202, respectively, in order orappearance).

FIGS. 16A-C show the recombinant M13 filamentous bacteriophage genomesequence used in the example that serves as the input to all Pythonscripts to generate the scaffold strand of the DNA nanotubes (SEQ ID NO:14).

FIGS. 17A-D list the sequences used in the example for front monomercore staples of a six-helix bundle DNA nanotube (SEQ ID NOS 15-182,respectively, in order or appearance).

FIGS. 18A-D list the sequences used in the example for rear monomer corestaples of a six-helix bundle DNA nanotube (SEQ ID NOS 203-370,respectively, in order or appearance).

FIG. 19 a shows a gel-shift analysis of folding and heterodimerizationof DNA nanotubes.

FIG. 19 b shows a negative-stain electron micrograph of DNA nanotubeheterodimers.

FIG. 19 c shows a photograph of the birefringence exhibited betweencrossed polarizers by DNA nanotube dimmers at 28 mg/mL in a glass NMRtube.

FIG. 19 d shows the NMR spectrum of a 90% H₂O/10% D₂O sample containing28 mg/mL DNA nanotube heterodimers.

FIG. 20 shows the ²H NMR spectra of D₂O in liquid crystalline DNAnanotubes recorded at 30 degrees C. and ¹H frequency of 500 MHz. Inpanel (A), the ²H quadrupolar coupling constant was 6.7 Hz forliquid-crystalline DNA nanotube at a concentration of 30 mg/ml in 50 mMHEPES, 50 mM NaCl, 10 mM MgCl₂, pH 7.5. In panel (B), 100 mM LMPGdetergent was added, yielding a coupling constant of 5.1 Hz (consistentwith mere dilution of the D₂O). Panel (C) shows that no change in thecoupling constant was observed 24 hours after addition of the detergent.

FIG. 21 shows analysis of DNA nanotubes. FIG. 21 a shows native agarosegel electrophoresis. I, 1 kb ladder; II, naked 7308 base scaffold; III,folded DNA nanotube. FIG. 21 b shows negative-stain electron micrographof 200 nm DNA nanotube; scale bar is 50 nm. FIG. 21 c shownegative-stain electron micrograph of 400 nm DNA nanotube, scale bar is200 nm.

FIGS. 22 a-f show negative-stain electron micrographs of DNA monomersand heterodimers. FIG. 22 a shows a DNA front monomer at 68000×magnification. FIG. 22 b shows a DNA nanotube heterodimer at 49000×magnification. FIG. 22 c shows DNA nanotube front monomers at 23000×magnification. FIG. 22 d shows DNA nanotube heterodimers at 18500×magnification. FIG. 22 e shows DNA nanotube front monomers at 6800×magnification. FIG. 22 f shows DNA nanotube heterodimers at 6800×magnification.

FIG. 23 a shows an analysis of the residual dipolar couplings (RDC's)measured for the detergent-reconstituted transmembrane domain of the ζchain of the T-cell receptor complex, weakly aligned in a 28 mg/mL DNAnanotube mixture. Shown is a 0.98 correlation coefficient between theobserved backbone RDC's and the RDC's predicted for the known NMRstructure of the ζ-ζ transmembrane domain (2HAC) obtained from theProtein Data Bank.

FIG. 23 b shows the principal axes of the alignment tensor relative to2HAC, the ζ-ζ dimer of the T cell receptor.

DETAILED DESCRIPTION Definitions

The term “nanotube” as used herein refers to a cylindrical arrangementof nucleic acid helices aligned in parallel and linked to one another,forming a tubular structure with approximate radial symmetry around acentral axis.

The term “scaffold nucleic acid” as used herein refers to asingle-stranded nucleic acid that is able to fold into variousconformations through the complementary binding of shortersingle-stranded nucleic acids (staple oligonucleotides) tonon-contiguous segments of the longer nucleic acid.

The term “staple oligonucleotide” as used herein refers to asingle-stranded oligonucleotide with successive segments that arecomplementary to non-contiguous segments of a scaffold, each scaffoldsegment forming part of a different helix in a nucleic acid nanotube. Asused herein, the term “staple” refers to staple oligonucleotide.

The term “crossover” as used herein refers to the point at which astaple oligonucleotide crosses over from a binding site on one helix toa binding site on an adjacent helix in a nucleic acid nanotube. Acrossover comprises either a covalent bond joining atoms in adjacenthelices or a chemical group which is covalently linked to atoms inadjacent helices. The chemical group can be, for example, a phosphategroup which forms part of the nucleic acid backbone of a stapleoligonucleotide.

The term “seam” as used herein refers to the point at which a scaffoldnucleic acid crosses from one helix to an adjacent helix. A seamcomprises either a covalent bond joining atoms in adjacent helices or achemical group which is covalently linked to atoms in adjacent helices.The chemical group can be, for example, a phosphate group which formspart of the nucleic acid backbone of a scaffold nucleic acid.

DESCRIPTION

The inventors have discovered how to make nucleic acid nanotubes of auniform length that will self-assemble into liquid crystals. A solutioncomprising liquid crystalline nucleic acid nanotubes is resistant todetergent and enables liquid-crystal NMR spectroscopy of membraneproteins solubilized in detergent. Rod-like molecules are more likely toself-assemble into liquid crystals if they have large aspect ratios(length-to-cross-section diameter) and if they are homogeneous inlength.

Nucleic acid nanotubes have been prepared using DNA origami techniques.These nanotubes can form detergent-resistant liquid crystals that makepossible the accurate measurement of NMR residual dipolar couplings(RDC's) for a wide array of detergent-solubilized proteins. Acquisitionof RDC's, which encode global orientation constraints, facilitates thede novo NMR structure determination of polytopic alpha-helical membraneprotein monomers larger than 15 kDa in size. The previous size limit forsolution-NMR-based de novo structure determination of membrane proteinscan be extended by employing liquid-crystalline nucleic acid nanotubesto facilitate the accurate measurement of residual dipolar couplings,from which global orientation information can be derived. Thus thesenanotube liquid crystals have made feasible the structure determinationof a wide range of biomedically important targets that currently arevery difficult to characterize.

Multi-helix bundle nucleic acid nanotubes were prepared by adapting thescaffolded DNA origami technique described by Rothemund (Rothemund, P.W., J. Biomol. Struct. Dyns. 22, addendum, 2005; and Rothemund, P. W.,Nature 440, 297-302, 2006; both hereby incorporated by reference intheir entireties). In one embodiment, the origami technique was used tocreate a six-helix bundle DNA-nanotube architecture similar to thatdescribed by Mathieu et. al (Nano Lett. 5, 661-5, 2005). The scaffoldDNA used to construct the multi-bundle DNA nanotubes can be one or morelong single-stranded DNA molecules of known sequence. To the scaffoldare added many short staple oligonucleotides with complementarity to atleast two sections of the scaffold DNA, the staple oligonucleotidesforce the scaffold into the shape of an array of parallel doublehelices. This construction technique permits the construction of bundlescomprising various numbers of helices, and of predictable lengths. Thelength of a bundle is determined by the length of the scaffold DNA,which is folded using the staple oligonucleotides into approximatelyequal smaller lengths of DNA helices that are linked to one another. Theaverage length of the nanotubes is given by the length of thesingle-stranded scaffold strand divided by the number of double-strandedhelices present in each nanotube. Each helix is linked to an adjacenthelix by at least two of the staple oligonucleotides which cross over toan adjacent helix. The bundles are linked together in this manner toform a closed tube-like structure. In a preferred embodiment of thisinvention, six-helix bundle DNA nanotubes were assembled by combining asingle-stranded scaffold DNA with a plurality of oligonucleotidescomplementary to segments of the scaffold, in a manner that causes thelength of the DNA nanotube to be one-sixth of the length of a DNA doublehelix comprising the scaffold as one strand. Thus, adapting the DNAorigami technique to the preparation of DNA nanotubes results innanotubes of predictable and uniform length and aspect ratio.

The nanotubes of the present invention are particularly well-suited toforming liquid crystals useful in a variety of applications. Moreover,the efficiency of producing a liquid crystal nanotube solution issubstantially improved with the present methods. Competition for bindingto the scaffold is likely to select for those oligonucleotides withfewer defects, thus mitigating somewhat complications from usingchemically-synthesized oligonucleotides. This strategy allows forflexibility in the length of each double helix in the array, as well asin the angle of curvature between any three parallel helices. The resultis a robust and facile method. This method does not require any sequencedesign for the scaffold, nor does it require purification of theoligonucleotides. Thus the amount of labor required for assembly of suchstructures is reduced greatly compared to previous methods, and thematerial costs are relatively low.

Sequence Structure of the Nucleic Acid Nanotubes

The basic strategy of preparing DNA origami structures is described byRothemund (Rothemund, P. W., J. Biomol. Struct. Dyns. 22, addendum,2005; and Rothemund, P. W., Nature 440, 297-302, 2006; both herebyincorporated by reference in their entireties). According to theinvention, one or more scaffold nucleic acids are combined with aplurality of staple oligonucleotides whose sequences are chosen to formcomplementary base pairings with the scaffold strand(s), thereby causingthe scaffold to fold into a framework which, together with thebase-paired staple oligonucleotides, forms three or more double heliceslinked side to side (i.e., a multi-helix bundle) to form a nanotube.

The double helices comprising each component of a multi-helix bundle canbe rendered as a sketch drawing, followed by conversion of the generalstructure into an Adobe Illustrator file that indicates the details ofthe spacing between scaffold crossovers and oligonucleotide crossovers.The minimum distance between scaffold crossovers and oligonucleotidecrossovers on adjacent lines is about 10 base pairs. In a preferredembodiment, the DNA nanotube structure mimics the shape and size of Pfl,a rod-like viral particle that is 6 nm in diameter and 2 μm in length.Its structural rigidity and negative-charge surface density allow it toform a stable and useful liquid crystal at low concentrations. Toachieve a Pfl-like DNA structure, a six-helix bundle DNA-nanotubearchitecture can be adopted. This design resembles a parallel array ofsix double helices for which every set of three adjacent helices framesa dihedral angle of 120 degrees (FIGS. 1 a and 1 d). Adjacent doublehelices are held together by Holliday-junction crossovers that occurevery 42 base pairs (FIG. 1 f). For each monomer, a 7308-base,M13-derived single-stranded circle of DNA (New England Biolabs) isemployed as a “scaffold” and 168 single strands of DNA of length 42bases, programmed with complementarity to three separate 14-base regionsof the scaffold, are employed as staple oligonucleotides (“staples”)(FIG. 1 f). The distance between scaffold crossovers and oligonucleotidecrossovers on adjacent lines is 42 base pairs, which results in goodscaffold folding kinetics and thermodynamics. The staples self-assemblewith the scaffold into the shape of six parallel double helices curledinto a tube.

Each pair of adjacent helices should have at least two crossovers inorder to enforce parallelism between the helices. The distance betweenoligonucleotide crossovers along a given line must be an even number ofhalf-turns, usually 32, 42 or 52 base pairs, leading to a pitch spacingof 10.7 base pairs, 10.5 base pairs, or 10.4 base pairs, respectively.The distance between scaffold and oligonucleotide crossovers must be anodd number of half-turns. For the 32 base pair spacing, this correspondsto distances of 16+16 and 5+27 base pairs. For the 42 base pair spacing,this corresponds to distances of 16+26 and 5+37 base pairs. For the 52base pair spacing, this corresponds to distances of 26+26, 16+36, and5+47 base pairs. In one embodiment, a five-helix bundle structurerequires a 108 degree angle between any three adjacent helices. At 10.8base pairs per turn, 14 base pairs yields 1.30 turns, yielding arotation along the helix of 360+108 degrees. In another embodiment, aseven-helix bundle structure requires a 128.5 degree angle between anythree adjacent helices. At 10.33 base pairs per turn, 14 base pairsyields 1.1355 turns, which is a rotation along the helix of 360+128degrees.

In a preferred embodiment, a six-helix bundle requires a 120 degreeangle between any three adjacent helices. With 42 base pairs betweencrossovers, the average twist of the helix is 10.5 base pairs per turn.At 10.5 base pairs per turn, 14 base pairs yields 1.33 turns, which is arotation along the helix of 360+120 degrees. There are 42 base pairsbetween colinear crossovers (crossovers to the same adjacent helix), andeither 14 or 28 crossovers along any helix to either adjacent helix.This implementation of the six-helix bundle uses oligonucleotides thatare all 42 bases long, and whose ends line up with the positions ofoligonucleotide crossovers on adjacent lines. This positioning isfavorable in that chemical moieties added to the ends of theoligonucleotides will extend out from the helix orthogonal to the convexsurface of the six-helix bundle.

In a six-bundle DNA nanotube, there are six DNA helices, and thescaffold is divided into six virtual strands. The top and bottom virtualstrands depicted in FIG. 2A are continuous fragments of the scaffold.The middle four virtual strands each are composed of two pieces of thescaffold strand, separated by the strand seam. A 7308 base scaffold DNAstrand results in virtual strands that are 7308/6 or 1218 bases long.Using a 42 base pair structure for the oligonucleotides, there are 29pseudo-repeats of the basic staple oligonucleotide structure. Thescaffold structure in the preferred embodiment is as follows, with thenumbers representing the relative values of the base pair positions:

-   -   0-1217: virtual strand 1    -   1218-1829: upstream component of virtual strand 2    -   1830-2455: downstream component of virtual strand 3    -   2456-3109: upstream component of virtual strand 4    -   3110-3725: downstream component of virtual strand 5    -   3726-4943: virtual strand 6    -   4944-5545: upstream component of virtual strand 5    -   5546-6109: downstream component of virtual strand 4    -   6110-6701: upstream component of virtual strand 3    -   6702-7303: downstream component of virtual strand 2

For helix 1, the first oligonucleotide strand attachment starts atposition 16 from the 5′ end of the virtual strand 1 at the proximal endof the nanotube, and binds a 14-base section of virtual strand 1 with a14-base section of virtual strand 2 and a 14-base section of virtualstrand 6. For helix 2, the first oligonucleotide strand attachmentstarts at position 26 from the 5′ end of virtual strand 2 at the distalend of the nanotube, and binds a 14-base section of virtual strand 1 toa 14-base section of virtual strand 2 and a 14 base section of virtualstrand 3. For helix 3, the first oligonucleotide strand attachmentstarts at position 2 from the 5′ end of virtual strand 3 at the proximalend of the nanotube, and binds a 14-base section of virtual strand 3 toa 14-base section of virtual strand 4 and a 14-base section of virtualstrand 5. For helix 4, the first oligonucleotide strand attachmentstarts at position 40 from the 5′ end of virtual strand 4 at the distalend of the nanotube, and binds a 14-base section of virtual strand 4 toa 14-base section of virtual strand 5 and a 14-base section of virtualstrand 6. Most of the staple oligonucleotides in this embodiment are 42base pairs long and attach to three non-contiguous sections of thescaffold DNA to produce the appropriate folding to generate thesix-helix bundle nanotube.

A monomer can be conceptualized as a series of 28 pseudo-repeatsegments, each consisting of six parallel double helices that are 42base pairs long, flanked by jagged overhangs on either end of the object(FIG. 1 b). Each segment can be conceptualized as a series of threesubsegments, for which every double helix is 14 base pairs long (FIG. 1f). Six of the twelve strands of a subsegment are provided by thescaffold strand, three are provided by one staple strand, and three byanother staple strand. Adjacent subsegments are related by 120-degreescrew pseudosymmetry. The scaffold generall_(y) does not cross overbetween helices, except for four times in the middle of each monomer toproduce a “seam”, and three times on each monomer end (FIG. 1 c).

DNA nanotube monomers can be multimerized using the appropriate designparameters. The inclusion of a seam in the design allows for the linkageof monomers in a head-to-tail fashion instead of in a head-to-headfashion, as is evident from consideration of the polarity of thescaffold strand within each double helix (FIG. 3 e). Three extra staplestrands block the head of the front monomer, and four extra staplestrands block the tail of the rear monomer (FIGS. 3 a and 3d). Tofacilitate heterodimerization, three extra staple strands with unpairedbases decorate the tail of the front monomer, and three extra staplestrands with unpaired bases decorate the head of the rear monomer (FIGS.3 b and 3c).

The model is then converted into DNA sequences; this can beaccomplished, for example, by coding performed by a Python program. Anexample is provided in FIGS. 4A-4D. The program performs the followingtasks: (1) input the scaffold strand sequence; (2) break the scaffoldstrand sequence into virtual strands corresponding to each paralleldouble helix; (3) break each virtual strand into complementary sequencetokens; and (4) generate the oligonucleotide sequences as catenatedtokens.

For nanotube heterodimerization, a computer program can be written togenerate staple strand sequences given the sequence of the scaffold (SeeFigures S3 a-c). A first Python script can be used to generate frontmonomer core oligonucleotides and head caps (FIGS. 5A-5B). A secondPython script can be used to generate rear monomer core oligonucleotidesand tail caps (FIGS. 6A-6B). A third Python script can be used togenerate front monomer tail connector oligonucleotides and rear headconnector oligonucleotides (FIGS. 7A-7B). Using two cyclic permutationsof the scaffold sequence as input to the program can generateindependent sets of staple-strand sequences for folding two differentmonomer nanotubes. Therefore copies of the same scaffold molecule can beused to generate two chemically-distinct species.

It is understood that many possible sequence combinations exist whichcan give rise to a given nanotube structure. The initial choice ofscaffold strand sequence will determine the sequences of the stapleoligonucleotides. However, once a particular scaffold strand is chosen,any given point along the sequence of the scaffold strand can be chosenas a starting point to build the nanotube structure. The choice ofscaffold sequence and starting point, together with the nanotubegeometry and the number and position of crossovers and seams, willdetermine the sequences of the staple oligonucleotides. Furthermore, thesequences of staple oligonucleotides can be optimized in order to avoidunintended binding that can give rise to defective structures or poorassembly kinetics.

Length of the DNA Nanotubes

The length of the DNA nanotubes is 50 nm or more. In one embodiment ofthe invention, the length of the DNA nanotubes is 200 nm or more. In apreferred embodiment, the length of the DNA nanotubes is about 400 nm. Alength of 400 nm can be achieved, for example, with a scaffold DNAstrand 7308 bases long folded into six strands to which complementarystaple oligonucleotides are bound, forming six-helix bundles.Preferably, the length of the nanotubes varies by no more than 20% ofthe average length of the nanotubes, and more preferably by no more than10% of the average length of the nanotubes. If a nanotube compriseshelices of different lengths, then the length of the longest helix isconsidered the length of the nanotube.

The lengths of the nanotubes formed using this technique can also bemodified through end-to-end multimerization of the bundle structures. Ina preferred embodiment of the invention, head-to-head and tail-to-tailmultimerization of the bundles can be generated from a scaffold DNAconfiguration in which the bends of the scaffold occur only at the endsof the bundles. In a more preferred embodiment of the invention,head-to-tail multimerization of the DNA bundles can be generated from ascaffold configuration in which some of the scaffold bends occur withinthe length of the DNA bundles, forming a seam across which the scaffoldDNA does not cross.

In a preferred embodiment, the virtual strands are connected to eachother by the staple oligonucleotides in a staggered manner. Virtualstrands 1 and 2 (see FIG. 2B) each have 16 base pairs available formultimerization on the proximal end of the nanotube, and 26 base pairsavailable for multimerization on the distal end. Virtual strands 3 and 4each have 2 base pairs available for multimerization on the proximal endof the nanotube, and 40 base pairs available for multimerization on thedistal end. Virtual strands 5 and 6 each have 40 base pairs availablefor multimerization on the proximal end of the nanotube, and 2 basepairs available for multimerization on the distal end. Thus theconnecting region for each multimerized strand is 42 bases long,maintaining a constant staple oligonucleotide length and allowing forhead-to-tail multimerization of the DNA nanotubes.

Dimerization of the DNA nanotubes can be achieved, for example, as shownin 3. FIG. 3 shows schematic views of the pre-dimerization monomers thatcan combine to form heterodimer DNA nanotubes. Specifically, FIGS. 3 a-3d show the scaffold-plus-staples schematic views of the front and rearoverhangs of the monomers. One strand of each double helix can becontributed by the scaffold (darker lines in FIGS. 3 a-3 e), and theother strand can be contributed by a staple oligonucleotide. Base pairsin the Figure are depicted as short vertical lines between the pairedstrands. Helices 1-6 are labeled in the center of FIGS. 3 a-b and 3 c-d.FIG. 3 a shows the front monomer head segment. Three staple strands canserve to cap the front monomer head (see DNA sequences, FIG. 8). FIG. 3b shows the front monomer tail segment, which has three staple strands(see DNA sequences, FIG. 9) with a total of 26 unpaired bases decoratingthe tail (2 bases in helix 2, 12 bases each in helices 5 and 6). Thescaffold strand in this region is unpaired for 36 bases (12 bases eachin helix 1, 3, and 4). FIG. 3 c shows the rear monomer head segment.Three staple strands (see DNA sequences, FIG. 10) on this portion of themonomer have a total of 36 unpaired bases decorating the head. Theseunpaired regions are complementary to the corresponding 36 unpairedbases of the front monomer tail scaffold strand. The 26 unpaired basesin the rear monomer head scaffold strand can be complementary to the 26unpaired bases of the three staple strands that decorate the frontmonomer tail. In the DNA nanotube heterodimer, these unpaired regionscan match up to form the complete intermonomer junction, as shown inFIG. 3 e. FIG. 3 d shows the rear monomer tail segment. Four staplestrands can serve to cap the rear monomer tail (see DNA sequences, FIG.11). FIG. 3 e shows the junction between the head and tail monomersforming the assembled heterodimer. The scaffold crossovers (darkervertical lines) that form an internal seam for each monomer occur atsegments 14 and 15, as shown in FIGS. 1 b and c.

In the nucleic acid nanotubes of the inventions, the scaffold strand isarranged with base pair sequences optimized to avoid unintended bindingevents between staple strands and the scaffold strand, or betweendifferent sections of the scaffold strand. The scaffold strand can bederived from a natural source whose base pair sequences have beencompletely characterized. In one embodiment, the scaffold strand isderived from the M13 mp 18 viral genome, which is well-characterized andrelatively inexpensive to generate in large quantities. It is alsoamenable to recombinant approaches to insert or delete sequences. Thescaffold strand can also be an entirely artificial sequence, a modifiednatural sequence, or any combination of natural and artificialsequences.

In another embodiment, plasmids based on the pBluescript vector can beused where a shorter, exact number of bases is desired. This avoidshaving extra unscaffolded material that may interfere with folding ofthe scaffold. With pBluescript, there is more flexibility with insertingDNA's that are many kilobases in size, without concern about plasmidinstability. To facilitate the excision of a single-strand DNA targetinsert from the generic vector, inverted repeat restriction sites can beintroduced into the vector. Inverted repeat EcoR I sites separated by 20base pairs can be added upstream of the target sequence. Inverted repeatHind III sites separated by 20 base pairs can also be added downstreamof the target sequence. In the single-stranded DNA, the repeated sitesfold up to form double-stranded sites that are recognizable by theappropriate restriction enzyme.

Assembly of the Nanotubes

The nucleic acid nanotubes of the present invention are self assembling.The scaffold strand and a molar excess of staple oligonucleotides areadded to a desired buffer, which preferably contains MgCl₂. The solutionis heated to a temperature sufficient to denature all the nucleic acidscontained therein (e.g., 90° C.), and then slowly allowed to cool. Thestep of cooling should be such that the solution returns to atemperature, e.g., room temperature, which permits assembly of thenanotubes over approximately 1 hour to 24 hours, e.g., over 2 hours or20 hours.

Example 1 Preparation of DNA Nanotubes

M13 single stranded scaffold DNA (sequence shown in FIGS. 12A-C) wasobtained from phage produced from infected F+bacteria grown in 2×YTmedia. Purified single-stranded DNA was extracted from the phage using aQiagen Gigaprep ion-exchange column. Six-helix bundle DNA nanotubes werefolded directly from the eluate of a Qiagen Gigaprep ion-exchangecolumn, eluted at 50 mM Tris pH 8.5 (Fisher Scientific), 1.6 M NaCl(Fisher Scientific), 15% isopropanol. In the folding reaction, thebuffer was diluted to 1 M NaCl, 9% isopropanol, along with 50 nM of theorganic chemical buffer HEPES(4-2-hydroxyethyl-1-piperazineethanesulfonic acid) pH 7.5 (Sigma), 10 mMMgCl₂ (Fisher Scientific). The scaffold concentration was at 6 nanomolarand the staple oligonucleotide (sequences shown in FIG. 13A-D)concentrations were at 36 nM each. The isopropanol did not interferewith the folding. Folding was performed by heating the suspension in 100mL Pyrex bottles in 2 L boiling water baths to 90° C., then covering thelid and allowing to cool to room temperature over the course of 20hours.

The folded six-helix bundle DNA nanotubes thus formed were separatedfrom the excess oligonucleotides by precipitation with 40% ethanol. TheDNA nanotubes survived desalting with a 75% ethanol wash followed bydehydration in a speedvac. After drying, the DNA nanotubes wereresuspended in a desired volume of buffer, without evidence ofaggregation or other misfolding.

Example 2 Recombinant M13 Bacteriophage Plasmid (p7308) Construction

Recombinant M13 filamentous bacteriophage was prepared by replacement ofthe BamHI-XbaI segment of M13 mp18 by a polymerase chainreaction-generated 59 base pair (bp) fragment encoding arandomly-selected sequence (FIG. 14), flanked by positions −25 to +25 ofthe middle of the XbaI cut site (T^CTAGA, or base 6258). A list ofoligodeoxyribonucleotides that were used to construct the insert withflanking regions (109 by total) is shown in FIG. 15. Double-stranded(replicative form) bacteriophage M13 DNA bearing the 59 base insert wasprepared as described in Sambrook, J. & Russell, D. Molecular cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 2001). The 59.bp insert was verified by a double restrictiondigest with BainHI and XbaI, followed by sequencing. The result was amodified bacteriophage M13 genome, 7308 bases in length. The fullsequence is shown in FIG. 16.

Example 3 Nanomole-Scale Production of M13 Bacteriophage Single-StrandedDNA

Recombinant M13 bacteriophage RF dsDNA was transformed into JM101 cellsand grown overnight at 37° C. on an LB-agar plate (BD Diagnostics). Asingle, well-isolated plaque was used to inoculate 2 ml of 2×YT mediumin a 14 mL sterile culture tube and agitated for 8 hours at 37° C.Bacterial cells were pelleted by centrifugation and phage was recoveredfrom the supernatant by polyethylene glycol fractionation (incubation onice for 30 minutes using a final concentration of 4% PEG8000, 0.5 MNaCl) followed by centrifugation. The phage was resuspended in 1000, of10 mM Tris.Cl pH 8.5 (Fisher Scientific) and labelled “pre-inoculationphage.” E Coli JM109 cells were grown overnight in 3 mL of 2×YT mediumat 37° C. The 3 mL of JM109 culture was added to a 2 L flask containing300 mL 2×YT medium supplemented with MgCl₂ to 5 mM final concentrationand incubated at 37° C. on a shaker at 300 rpm. When the bacterialculture reached A₆₀₀=0.5, 50 μL of the “pre-inoculation phage” stock wasadded. The infected culture was grown at 37° C., shaking at 300 rpm foran additional 4 hours. Phage was recovered as described above, andresuspended in 3 mL 10 mM Tris.Cl pH 8.5 and labelled “inoculationphage.” Titer of “inoculation phage” was measured by plating out serialdilutions using saturated JM109 culture and LB-top agar plates. Titer ofJM109 cells at A600=0.5 was measured by plating out serial dilutions onLB-agar plates. For nanomole-scale production of phage, twelve 2 Lflasks each containing 300 mL 2×YT medium supplemented with 5 mM MgCl₂,were inoculated with 3 mL overnight JM109 culture and incubated at 37°C. shaking at 300 rpm. When density reached A₆₀₀=0.5, each flask wasinfected with “inoculation phage” at an MOI=1. Phage was harvested asdescribed, and resuspended in 0.5% of the original culture volume in 10mM Tris.Cl pH 8.5. Single-stranded DNA was isolated from phage byalkaline/detergent denaturation as follows: Two volumes of lysis buffer(0.2 M NaOH, 1% SDS) were added to the resuspended phage, followed by1.5 volumes neutralization buffer (3 M KOAc pH 5.5). Lysed phage wascentrifuged for 10 minutes at 16000 ref. The supernatant was combinedwith one volume of 200 proof ethanol and centrifuged for 10 minutes at16000 ref. Pelleted ssDNA was washed twice with 75% ethanol,centrifuged, and resuspended in 5% of the original culture volume in 10mM Tris.Cl pH 8.5. The concentration of the recovered ssDNA wasestimated on a UV/visible spectrophotometer (Beckman coulter) using anextinction coefficient=37.5 μg/mL for A₂₆₀=1.

Example 4 Preparation of 800 Nm DNA Nanotube Heterodimers

A detergent-resistant liquid crystal of 0.8 μm-long DNA nanotubes hasbeen designed and constructed, and has been shown to induce weakalignment of membrane proteins. The nanotubes are heterodimers of0.4-μm-long six-helix bundles each self-assembled from a 7.3-kilobasescaffold strand and over 170 short oligonucleotide staple strands.Desalted DNA oligonucleotides, normalized by concentrations to 150 μM,were purchased from Invitrogen (see DNA sequences of FIGS. 8-11, 17,18). Equal volumes of each staple oligonucleotide were pooled intogroups: front monomer staple stock (DNA sequences of FIGS. 8, 9, 17) andrear monomer staple stock (DNA sequences of FIG. 10, 11, 18). The frontmonomer staple stock includes front head cap staples (DNA sequences ofFIG. 8), front core staples (DNA sequences of FIG. 17), and front tailconnector staples (DNA sequences of FIG. 9). The rear monomer staplestock includes rear head connector staples (DNA sequences of FIG. 10),rear core staples (DNA sequences of FIG. 18), and rear tail cap staples(DNA sequences of FIG. 11). Concentrations of the pooled staple stockswere estimated on a UV/visible spectrophotometer using an extinctioncoefficient=33 μg/mL for A₂₆₀=1.

Front and rear monomers were prepared with their respective staplestocks, but otherwise using an identical protocol. The front monomerfolding mixture was prepared by combining p7308 ssDNA (30 nM), frontmonomer staple stock (300 nM each staple strand), 50 mM HEPES pH 7.5, 50mM NaCl, and 30 mM MgCl₂ in a final volume of 76.8 mL. The foldingmixture was aliquoted into four 96-well plates (Molecular BioProducts)(200 μl, per well), and folded on a thermal cycler (MJ Research Tetrad)with the following program:

1. 80° C. for 5:00

2. 80° C. for 2:00 (−1° C. per cycle)

3. Go to 2, 60 times

4. End

Folded material was pooled into a 250 mL polypropylene centrifugebottle. Folded nanotubes were separated from excess staple strands viaPEG fractionation as follows: 19.2 mL of 20% PEG8000 (FisherScientific), 2.5 M NaCl was added to mixture, which was then centrifugedat 15000 rcf for 15 minutes. The supernatant was discarded, and thenanotube pellet was resuspended in 38.4 mL 50 mM HEPES pH 7.5, 50 mMNaCl, and 30 mM MgCl₂. A second PEG fractionation was carried out asfollows: 9.6 mL of 20% PEG8000, 2.5 M NaCl was added to mixture, whichwas then centrifuged at 15000 rcf for 15 minutes. The supernatant wasdiscarded, and the nanotube pellet was resuspended in 38.4 mL 50 mMHEPES pH 7.5, 50 mM NaCl, and 30 mM MgCl₂.

Nanotube heterodimers were formed by combining front and rear monomermixtures together and incubating at 37° C. for 2 hours. Two volumes ofequilibration buffer (750 mM NaCl, 50 mM MOPS, pH 7.0, 15% isopropanol,0.15% Triton X-100 (v/v)) were added to the mixture. Heterodimers wereloaded on a Qiagen-Tip 10000 gravity-flow ion-exchange columnequilibrated with 75 mL equilibration buffer. The column was washed withsix 100 mL volumes of 1 M NaCl, 50 mM MOPS, pH 7.0, 15% isopropanol(v/v). Then the nanotubes were precipitated by addition of one volume200 proof ethanol, centrifuged at 15000 rcf for 15 minutes, washed twicewith 75% ethanol, and resuspended in 3 mL 1 mM NaH₂PO₄ pH 7.0, 1 mMMgCl₂. The nanotube concentration was estimated via UV absorbance at 260nM assuming an extinction coefficient of A₂₆₀=1 for 50 μg/ml. Thenanotube heterodimer mixture was then concentrated by Speedvac vacuumcentrifugation to a final volume of 3004. Front and rear monomers werefolded in separate chambers via heat denaturation followed by coolingfor renaturation.

The front and rear monomers were mixed to self-assemble heterodimers(FIGS. 1 a-c, and 1 e). The joining of the tail of the front monomer tothe head of the rear monomer should generate a 42-base-pairpseudo-repeat segment (FIG. 1 e). In this segment, all six staplestrands bridge the two scaffolds, although by varying numbers of basepairs. In total, a net 62 base pairs must be broken to sever the linkagebetween successfully heterodimerized monomers.

Example 5 Demonstration of the Liquid Crystallinity of DNA Nanotubes

Six helix bundle DNA nanotubes from Example 1 were incubated in 25%ethanol, causing selective precipitation of the nanotubes, and leavingbehind the excess unbound staple oligonucleotides. Nine milligrams ofthe DNA nanotubes were resuspended in a volume of 6 mL 2.5 mM HEPES pH7.5, 2.5 mM NaCl, 0.5 mM MgCl₂, and then dehydrated in a Savant speedvacconcentration system to achieve a final concentration of 30 mg/ml (300μL of a 3% suspension) and final buffer concentrations of 50 mM HEPES pH7.5, 50 mM NaCl, and 10 mM MgCl₂.

The liquid crystallinity of the DNA nanotube suspension in an NMR tubewas verified by observation of birefringence under crossed polarizers. Alow-salt, aqueous suspension of DNA-nanotube heterodimers at aconcentration of 28 mg mL⁻¹ forms a stable liquid crystal, as indicatedby strong birefringence observed through crossed polarizers, as shown inFIG. 19 c. (FIG. 19 c). The liquid crystals were diluted by 10% withdeuterated water, and were aligned for three hours in a 600 MHz NMRspectrometer. Strong birefringence was observed when the sample tube wasplaced at 45 degrees to the crossed polarizers.

Further evidence for liquid crystallinity of the DNA nanotube monomerswas obtained by NMR spectroscopy, measuring quadrupolar splitting of thedeuterium, where a coupling constant of 6.7 Hz was observed (FIG. 20A).Next, 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)](LMPG) detergent was added to 100 mM. After addition of the detergent,the coupling constant dropped to 5.1 Hz, which is consistent with the16% dilution of D₂O in the sample upon addition of the detergentsuspension. The liquid crystals remain stable over at least 24 hours inthe presence of the detergent (FIG. 20C).

When the suspension of 800 nanometer heterodimers is aligned in an 11.4Tesla magnetic field in the presence of 10% D₂O, the weakly-oriented HDOyields ²H quadrupolar splitting of 5.56 Hz (FIG. 19 d). The 1D ²Hspectrum shown in FIG. 19 d was obtained from a 10 mM NaH₂PO₄, 10 mMMgCl₂, 90% H₂O/10% D₂O sample containing 28 mg/mL DNA nanotubeheterodimers. NMR spectra were processed and analyzed using NMRPipe.Fitting of the dipolar couplings to the known ζ-ζ homodimer structurewas done by singular value decomposition (SVD), using the program PALES.The goodness of fit was assessed by both Pearson correlation coefficient(r) and the quality factor (Q).

Example 6 Characterization of DNA Nanotubes

Folded DNA nanotubes were analyzed using agarose gel electrophoresis andnegative-stain electron microscopy using uranyl formate (Pfaltz & Bauer)as the stain. Gel electrophoresis experiments indicated that themajority of scaffold molecules are folded as monomers, as they produceda single band upon agarose gel electrophoresis in the presence of 10 mMMgCl₂ (FIG. 21 a). Further analysis of folding and heterodimerization ofDNA nanotubes was conducted via electrophoresis in a 2% agarose gelcontaining 11 mM MgCl₂, 0.5 μG/mL ethidium bromide, 45 mM Tris base, 45mM boric acid, and 1 mM EDTA (pH 8.0), and is shown in FIG. 19 a. Themajority of DNA objects migrate as a single band in agarose-gelelectrophoresis (FIG. 19 a). This population presumably representswell-formed nanotube monomers, while slower migrating species apparenton the gel presumably represent misfolded or multimerized structures.Lane M is the marker lane with DNA size standards denoted by number ofbase pairs shown to the left of the lane. Lane 1 shows the M13-derivedsingle-stranded DNA scaffold. Lanes 2 and 3 show the front and rear DNAmonomers (including scaffold plus staples). Lanes 4 and 5 show the frontand rear monomers after PEG fractionation. Agarose-gel electrophoresisof heterodimers assembled from the two monomers indicates that themajority of DNA objects migrate as a single band (FIG. 19 a, Lane 6),although some misfolded objects are evident, as are a small populationof monomeric nanotubes. Lane 6 shows the heterodimers after a two-hourincubation of mixed monomers at 37° C.

Electron micrograph analysis was carried out using Image SXM. Thelengths of 20 well-isolated DNA nanotube monomers and 20 well-isolatedDNA nanotube dimers in several separate electron micrographs weremeasured manually using the segmented-line tool. Following thedimerization step, DNA nanotube dimers were diluted to 1 nMconcentration and prepared for imaging by negative stain with 0.7%uranyl formate (Pfaltz & Bauer) as previously described. (Ohi, M.,Cheng, Y., Walz, T. Biol. Proc. Online 6, 23-24 (2004)). Gilder FineBarGrids, 400 mesh, 3.05 mm O.D. (Ted Pella) were used. Imaging wasperformed on a Tecnai G² Spirit BioTWIN. Electron microscopy experiments(FIG. 21 b, 21 c) showed that the DNA nanotubes are much more rigid thandouble helices. If double helices are assumed to be 2 nm wide and 0.34nm per basepair, then the predicted width would be 6 nm, and thepredicted length would be either 200 nm or 414 nm. The length and widthof the imaged objects approximately matched the predicted dimensions.The DNA nanotube heterodimer mixture was also analyzed usingnegative-stain electron microscopy, and the results are consistent witha large fraction of intact nanotubes of length 402±6 nanometers (FiguresS2, a, c, and e). This measured length is in good agreement with thepredicted length of 400 nanometres for 28 segments that are 42 basepairs long, assuming a rise of 0.34 nanometers per base pair.Negative-stain electron microscopy also revealed nanotubes of length813±9 nanometers, as shown in FIG. 19 b (scale bar=500 nanometers) andFigures S2, b, d, and f). This measured length agrees well with thepredicted length of 814 nanometers for 57 segments that are 42 basepairs long.

Example 7 Solution NMR Methodology for Membrane-Protein StructuralDetermination

Membrane proteins play important roles in cell biology and medicine. Forexample, over half of hormones and neurotransmitters studied to datetransduce signals through members of the G-Protein Coupled Receptor(GPCR) family of membrane proteins. Similarly, over half of allcommercial drugs target GPCR's. Despite their importance, structures ofonly ˜100 membranes proteins have been solved to dateblanco.biomol.uci.edu/Membrane_Proteins_xtal.html holds a tally that isupdated regularly). The slow rate of membrane-protein structuredetermination represents a significant bottleneck for both basic andapplied bioscience discovery. This bottleneck largely derives fromdifficulties in forming well-ordered three-dimensional crystals ofmembrane proteins (Caffey M, Membrane protein crystallization, J.Struct. Biol. 142, 108-132, 2003). Solution NMR presents apotentially-attractive alternative for the study of many membraneproteins, as high-resolution structural information can be obtained forsystems up to 80 kD in size without the need for crystallization.

Solution NMR has advanced to the point where structure determination of30-kD water-soluble proteins has become routine. This has not been thecase, however, for membrane proteins. For solution NMR, macromoleculesmust be solubilized in water to facilitate fast tumbling; the faster thetumbling, the better the spectra. To promote water solubility, membraneproteins must be complexed with detergent micelles. The micelle-proteincomplex is considerably larger than the protein alone, and tumbling isrelatively slow as a result. This increase in effective size isespecially problematic for a-helical membrane proteins greater than 15kDa in size, where resonance peaks are closely spaced and becomeunresolvable with the fast coherence relaxation of the slowly-tumblingmacromolecules. Some of the larger alpha-helical membrane proteins whosestructures has been solved by solution NMR include the Misticmembrane-surface-associating protein (13 kDa) (Roosild T P, Greenwald J,Vega M, Castronovo S, Riek R, Choe S, NMR structure of Mistic, amembrane-integrating protein for membrane protein expression, Science307, 1317-1321, 2005) and subunit c of the ATP synthase (7 kDa) (GirvinM E, Rastogi V K, Abildgaard F, Markley J L, Fillingame R H, Solutionstructure of the transmembrane H+-transporting subunit c of the FIFO ATPsynthase, Biochemistry 37, 8817-8824, 1998). Recently, our collaboratorsin Dr. Chou's laboratory have used solution NMR for the de novostructure determination of the phospholamban pentamer, a 30-kDchannel-like protein that spans the sarcoplasmic reticulum membrane(Oxenoid K, Chou J J, The structure of phospholamban pentamer reveals achannel-like architecture in membranes, Proc Nat'l Acad Sci USA 102,10870-10875, 2005). In that case, however, the NMR spectra weresimplified because of the five-fold rotational symmetry in the complex.

For conventional NMR spectroscopy, the Nuclear Overhauser Effect (NOE)provides the only experimentally-measurable distance restraint fortertiary structure determination. Successful structure determinationrequires the correct assignment of most of the proton resonances, ademand that can be almost impossible to meet for poorly-resolved spectrasuch as those recorded for a-helical membrane proteins. Furthermore,NOE's only are detectable for distances shorter than five angstroms,thus determination of the global shape of extended proteins is subjectto compounded errors.

RDC's encode global orientational constraints that enable structuredetermination with only limited NOE assignments required. If a largenumber of accurate RDC's can be measured, then a full analysis of theNOESY spectra—which may in practice be unobtainable—becomes unnecessary.In this case, it will be sufficient to measure NOE's after selectivelabeling of amino acids, which simplifies the spectrum, or to measuresemi-quantitative distance constraints from paramagnetic-broadeningtechniques.

Residual dipolar coupling leads to informative resonance frequencysplitting. In the presence of an external field B that points in thez-direction, the z-component of the magnetic field from nucleus S willchange the magnetic field at I such that the resonance frequency of Iwill shift by a quantity that depends on the internuclear distance andon the internuclear angle with respect to the z-axis. If the protein isundergoing rapid isotropic tumbling, then the average perturbationaverages to zero.

In order to obtain information about the internuclear angles, then, eachprotein must be made to tumble in a weakly-ordered regime. Too muchordering and dipolar couplings become so strong that peaks areunresolvable, while too little ordering leads to undetectable levels ofdipolar coupling. The appropriate weak ordering, about 0.1%, can beachieved by dissolving the protein in the right concentration of asuitable alignment material. For example, water-soluble proteins can bealigned weakly by the required amount with ˜1.5-2% Pfl filamentousphage, which forms a liquid crystal at that concentration.

Membrane proteins can be weakly aligned. The easiest method forweak-alignment of proteins is through mixing the protein with aliquid-crystalline medium, such as Pfl filamentous phage, DMPC/DHPCbicelles, C12E5 polyethylene glycol, or cellulose crystallites. However,none of these media are compatible with detergent-solubilized membraneproteins. The only method currently available for weak alignment ofmembrane proteins involves the use of radially-compressed polyacrylamidegels (Oxenoid K, Chou J J, The structure of phospholamban pentamerreveals a channel-like architecture in membranes, Proc Nat'l Acad SciUSA 102, 10870-10875, 2005; Chou J J, Gaemers S, Howder B, Louis J M,Bax A, A simple apparatus for generating stretched polyacrylamide gels,yielding uniform alignment of proteins and detergent micelles, J BiomolNMR 21, 377-382, 2001; Chou J J, Kaufman J D, Stahl S J, Wingfield P T,Bax A, Micelle-induced curvature in a water-insoluble HIV-1 Env peptiderevealed by NMR dipolar coupling measurement in stretched polyacrylamidegel, J Am Chem. Soc 20, 2450-2451, 2002; Tycko R, Solid-state NMR as aprobe of amyloid fibril structure, Curr Opin Chem. Biol 4, 500-506,2000). A technical problem encountered during the weak alignment ofphospholamban was that the maximum protein concentration obtainable inthe gel was ˜0.2 mM, despite soaking in a solution with a proteinconcentration of 1-2 mM. Because of the low concentration, thesignal-to-noise ratio of the NMR signals was low. Long data acquisitiontimes were required, and the resultant RDC measurements were of limitedaccuracy. The difficulty in soaking detergent-solubilized membraneproteins into polyacrylamide gels is a well-known problem in the NMRcommunity.

The six-helix bundle DNA nanotubes described herein represent adetergent-resistant shape mimetic of the Pfl filamentous phage. TheseDNA nanotubes have similar liquid-crystalline behavior as Pfl, but arecompletely resistant to strong detergents such as SDS.

Example 8 Use of DNA Nanotube Liquid Crystal to Measure Backbone RDC'sfor the Transmembrane Domain of the T-Cell Receptor

All NMR experiments were performed on Bruker spectrometers equipped withcryogenic TXI probes at 30° C. The RDC's were obtained from subtractingJ or J+D couplings of the aligned sample from that of unaligned sample.The ¹H-¹⁵N RDC's were obtained from ¹J_(NH)/2 and (1J_(NH)+¹D_(NH))/2,which were measured at 600 MHz (1H frequency) by interleaving a regulargradient-enhanced HSQC and a gradient-selected TROSY, both acquired with80 ms of ¹⁵N evolution. The ¹⁵H_(α)-¹³C_(α) RDC's (1D_(CαHα)) weremeasured at 500 MHz (1H frequency) using a 2D CACONH quantitative¹J_(CαHα), experiment with interleaved spectra recorded at ¹J_(CαHα),modulation times of 1.83, 3.63, and 7.12 ms. This experiment wasmodified from the 3D CBCACONH quantitative J_(CH) experiment³⁰ usedprimarily for measuring protein side-chain ¹H_(β)-¹³C_(β) RDC's. TheCACONH was optimized for measuring the backbone ¹H_(α)-¹³C_(α) RDC'sonly. Since the ζ-ζ transmembrane (TM) domain is a homodimer obeyingtwo-fold rotational symmetry, the same RDC's are assigned to bothsubunits. The frequency labeled dimensions in this experiment are ¹H^(N)(direct) and ¹⁵N (indirect).

The DNA-nanotube liquid crystal enables the accurate measurement ofbackbone N_(H) and C_(α)H_(α) RDC's for the detergent-reconstituted ζ-ζtransmembrane domain of the T-cell receptor. The measured RDC's validatethe high-resolution structure of this transmembrane dimer. The DNAheterodimer nanotubes were tested for weak alignment of thetransmembrane (TM) domain (residue 7-39) of the ζ-ζ chain of the T-cellreceptor complex reconstituted in mixed dodecylphosphocholine(DPC)/sodium dodecyl sulfate (SDS) detergent micelles. The measured¹H-15N and ¹H_(α)-¹³C_(α) RDC's agree very well with the known NMRstructure of the ζ-ζ TM domain, with a correlation coefficient of theSingular Value Decomposition (SVD) fit, R_(SVD), of 0.98, or a freequality factor, Q_(free) 16% (FIG. 23 a). The magnitude of the alignmenttensor, D_(a), is −9.9 Hz (normalized to D_(NH)), which is ideal for RDCmeasurement and structure calculation. In addition, the axis of C₂rotational symmetry of ζ-ζ is parallel to the largest principal axis,A_(zz), of the alignment tensor (FIG. 23 b). This result is expectedfrom the rotational averaging of the dimeric complex around its C₂ axisin the aligned medium.

1. A method of performing NMR spectroscopy of membrane proteins usingnucleic acid nanotubes comprising the steps of: (a) suspending nucleicacid nanotubes in a solution that comprises a detergent; (b) forming aliquid crystalline phase comprising the nucleic acid nanotubes; (c)adding a membrane protein to the solution; and (d) performing NMRspectroscopy on the protein and nucleic acid nanotube mixture.
 2. Themethod of claim 1, wherein the membrane protein is present at aconcentration of at least 0.1 mM.